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This Provisional PDF corresponds to the article as it appeared upon acceptance. Fully formatted PDF and full text (HTML) versions will be made available soon. Thrombelastography and biomarker profiles in acute coagulopathy of trauma: A prospective study Scandinavian Journal of Trauma, Resuscitation and Emergency Medicine 2011, 19:64 doi:10.1186/1757-7241-19-64 Sisse R Ostrowski (sisse.ostrowski@gmail.com) Anne Marie Sorensen (anne.marie.01.soerensen@rh.regionh.dk) Claus F Larsen (claus.falck.larsen@rh.region.dk) Par I Johansson (per.johansson@rh.regionh.dk) ISSN 1757-7241 Article type Original research Submission date 9 September 2011 Acceptance date 26 October 2011 Publication date 26 October 2011 Article URL http://www.sjtrem.com/content/19/1/64 This peer-reviewed article was published immediately upon acceptance. It can be downloaded, printed and distributed freely for any purposes (see copyright notice below). Articles in SJTREM are listed in PubMed and archived at PubMed Central. 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This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 1 Thrombelastography and biomarker profiles in acute coagulopathy of trauma: A prospective study Sisse R Ostrowski 1 , Anne Marie Sørensen 2,3 , Claus F Larsen 3 , Pär I Johansson 1 SRO: sisse.ostrowski@gmail.com AMS: anne.marie.01.soerensen@rh.regionh.dk CFL: claus.falck.larsen@rh.region.dk PIJ: per.johansson@rh.regionh.dk 1 Section for Transfusion Medicine, Capital Region Blood Bank, Copenhagen University Hospital, Rigshospitalet, Blegdamsvej 9, DK-2100 Copenhagen, Denmark. 2 Department of Anesthesia, Copenhagen University Hospital, Rigshospitalet, Blegdamsvej 9, DK-2100 Copenhagen, Denmark. 3 The Trauma Centre, Centre of Head and Orthopedics, Copenhagen University Hospital, Rigshospitalet, Blegdamsvej 9, DK-2100 Copenhagen, Denmark Correspondence and reprints Sisse R. Ostrowski MD PhD DMSc, Section for Transfusion Medicine, Capital Region Blood Bank, Copenhagen University Hospital, Rigshospitalet, Blegdamsvej 9, DK-2100 Copenhagen, Denmark. Tel.: +45 24430464; Fax +45 35390038; E-mail: sisse.ostrowski@gmail.com 2 Abstract Background Severe injury induces an acute coagulopathy associated with increased mortality. This study compared the Thrombelastography (TEG) and biomarker profiles upon admission in trauma patients. Methods Prospective observational study of 80 trauma patients admitted to a Level I Trauma Centre. Data on demography, biochemistry including standard coagulation tests, hematology, transfusions, Injury Severity Score (ISS) and TEG were recorded. Retrospective analysis of thawed plasma/serum for biomarkers reflecting tissue injury (histone-complexed DNA fragments), sympathoadrenal activation (adrenaline, noradrenaline), coagulation activation/inhibition and fibrinolysis (sCD40L, protein C, activated Protein C, tissue-type plasminogen activator, plasminogen activator inhibitor-1, D-dimer, prothrombinfragment 1+2, plasmin/α 2 - antiplasmin complex, thrombin/antithrombin complex, tissue factor pathway inhibitor, antithrombin, von willebrand factor, factor XIII). Comparison of patients stratified according to ISS/TEG maximum clot strength. Linear regression analysis of variables associated with clot strength. Results Trauma patients had normal (86%), hypercoagulable (11%) or hypocoagulable (1%) TEG clot strength; one had primary hyperfibrinolysis. Hypercoagulable patients had higher age, fibrinogen and platelet count (all p<0.05), none had increased activated partial thromboplastin time (APTT) or international normalized ratio (INR) and none required massive transfusion (>10 red blood cells the initial 24h). Patients with normal or hypercoagulable TEG clot strength had comparable biomarker profiles, but the few patients with hypocoagulable TEG clot strength and/or hyperfibrinolysis had very different biomarker profiles. Increasing ISS was associated with higher levels of catecholamines, histone-complexed DNA fragments, sCD40L, activated protein C and D-dimer and reduced levels of non-activated protein C, antithrombin, fibrinogen and factor XIII (all p<0.05). Fibrinogen and platelet count were associated independently with clot 3 strength in patients with ISS≤26 whereas only fibrinogen was associated independently with clot strength in patients with ISS>26. In patients with ISS>26, adrenaline and sCD40L were independently negatively associated with clot strength. Conclusions Trauma patients displayed different coagulopathies by TEG and variables independently associated with clot strength changed with ISS. In the highest ISS group, adrenaline and sCD40L were independently negatively associated with clot strength indicating that these may contribute to acute coagulopathy. Key words Trauma, coagulopathy, trauma induced coagulopathy (TIC), Thrombelastography (TEG), platelets, fibrinogen, FXIII, sympathoadrenal activation, sCD40L 4 Background Many severely injured patients develop an acute coagulopathy of trauma (ACT) already at the scene of the accident [1;2] and 25-35% are coagulopathic upon admission, a condition associated with a four-fold increase in mortality [3]. Previous studies have defined ACT as increases in plasma based coagulation tests (activated partial thromboplastin time (APTT), partial thrombin time (PTT), prothrombin time (PT), international normalized ratio (INR)) [4] but there is emerging evidence that the viscoelastic whole blood tests, Thrombelastography (TEG) and Rotation Thromboelastometry (ROTEM), can detect and discriminate between different types of traumatic coagulopathy [5] since this entity appears to change from normal to hypercoagulability, hypocoagulability and finally hyperfibrinolysis with increasing injury severity [1;6-11]. Immediate identification of the specific type of traumatic coagulopathy by TEG/ROTEM is of critical importance in order to goal-direct transfusion therapy and e.g. administer plasma, platelets, fibrinogen and/or antifibrinolytics to patients with evident hypocoagulability and/or hyperfibrinolysis [12;13]. We have used TEG to monitor hemostasis and guide transfusion therapy in massively bleeding patients since 2004 and this has significantly improved survival in these patients [14]. To our knowledge, no studies have directly compared outcomes in ratio-driven vs. TEG guided resuscitated bleeding trauma patients, and in addition to our previous finding of improved survival in bleeding patients resuscitated goal-directed according to TEG [14] we recently reported, in a meta-analysis of 16 studies of massively bleeding trauma patients, that the highest ratio of FFP and/or PLT to RBC was associated with a significantly reduced mortality (OR 0.49 (95% CI 0.43-0.57), p<0.0001) as compared to the lowest ratio [15]. TEG/ROTEM were recently recommended internationally as gold standard point-of-care tests in bleeding trauma patients [16;17]. Though the exact pathophysiologic mechanism(s) of the ACT are unclear, retrospective analyses of circulating biomarkers have pointed to downstream effects of tissue injury, sympathoadrenal activation and hypovolemic/hemorrhagic shock as drivers [3;4;18] of an enhanced early protein C (PC) activation, hyperfibrinolysis [3;4;18] and endothelial damage [19], which may all contribute to the coagulopathy. Despite 5 the recognized differences in presenting TEG/ROTEM profile in trauma patients [5], no studies have so far reported on circulating biomarker levels of tissue injury, sympathoadrenal activation and coagulopathy in trauma patients with different TEG profiles. The primary aim of this study was to investigate biomarkers of sympathoadrenal activation, tissue injury, coagulation activation/inhibition and fibrinolysis in trauma patients stratified according to injury severity and TEG profile upon admission. A secondary aim was to identify biomarkers independently associated with TEG maximum clot strength as this parameter is the parameter most strongly associated with bleeding, transfusion requirements and outcome in massively bleeding patients [5]. We hypothesized that progressive coagulopathy by TEG (from normal to hypercoagulability, hypocoagulability and hyperfibrinolysis) would be accompanied by evidence of increased sympathoadrenal activation, tissue injury, PC activation and hyperfibrinolysis [20]. Methods Study Design Prospective observational cohort study of trauma patients admitted directly to a Level I Trauma Centre (TC) at a tertiary hospital (Rigshospitalet, Copenhagen, Denmark, covering 2.5 million inhabitants) between March 2010 and November 2010. The study is part of an ongoing larger multicentre study [21], Activation of Coagulation and Inflammation after Trauma 3 (ACIT3), approved by the Regional Ethics Committee (H-4-2009-139), the Danish Data Protection Agency and conducted in accordance with the 2nd Declaration of Helsinki. Written informed consent was obtained from the patients or next of kin. Here we report on preliminary findings related to a cohort of 80 patients recruited to the ACIT3 study. 6 Patient selection ACIT3 study inclusions: Adult trauma patients (≥18 years) who met criteria for full trauma team activation and had an arterial cannula inserted. The latter was chosen since only patients with expected severe injuries have an arterial cannula placed immediately upon TC admission. Exclusion criteria, according to the multicentre study protocol [21], were arrival in the TC >2 hours after injury; >2,000 ml of intravenous fluids administered before hospital arrival; transfer from another hospital or burns >5% total body surface area. Patients were retrospectively excluded if they were taking anticoagulant/antiplatelet medications (except aspirin); had moderate or severe liver disease or had known bleeding diathesis. The 80 included patients were selected from the first 100 patients recruited to the ACIT3 study with complete data. We intended to include 80 patients because we measured an extensive number of biomarkers by ELISA, with each ELISA kit providing analysis of 80 samples. We aimed at including the most severely injured and/or coagulopathic patients and selected the 80 patients according to: Outcome (mortality or ICU admission post trauma; yes), transfusion of RBC within 6 hours (yes), RTS (<5.00, we had not access to ISS before later in the study period) or coagulopathy (APTT ≥35 sec, INR ≥1.2, Ly30 >1%/Cl30<95%; yes). This yielded 70 severely injured/coagulopathic patients, and additionally 10 patients (age 48 years (IQR 43-52), 60% males) were selected blinded from the remaining 30 patients to match their age and gender (see Table 1 for details on demography, injury severity etc.). The 20 patients not included in this study, had, compared to the included patients, comparable age and gender (41 years (IQR 33-53), 40% males) and APTT (26 (IQR 23-27), NS) but had, as expected, lower ISS (4 (IQR 2-10), p<0.001), mortality (0%, p=0.037) and INR (1.1 (IQR 1.0-1.1), p=0.007). Two of the 20 patients not included had a hypercoagulable TEG (MA>69, 10%). Data on demography, clinical and biochemical parameters, investigations, management and 30-day mortality were recorded and ISS scores were obtained from the Trauma Audit & Research Network (TARN) database. No patients received Tranexamic acid or catecholamines (Adrenaline or Noradrenaline) for hemodynamic stabilization prior to blood sampling. 7 Blood sampling Blood was sampled immediately upon arrival for standard arterial blood gas (ABG, Radiometer ABL 725/735, Copenhagen, Denmark), routine biochemistry and research analyses (citrate, heparin, EDTA plasma, serum). Routine biochemistry samples were analyzed in a DS/EN ISO 15189 standardized laboratory by a Sysmex XE- 2100 (hemoblobin, platelets, leukocytes) and ACL TOP (APTT, INR, AT, fibrinogen). Samples for functional hemostatic assays were kept at room temperature (RT) until analyzed precisely 1 h after sampling. Plasma samples were ice-cooled immediately whereas serum samples were kept at RT for 1 h before centrifugation (one (serum) or two (plasma) times 1800g at 5 °C for 10 min) and storage at -80 °C. Enzyme linked immunosorbent assay (ELISA) measurements Soluble biomarkers of tissue injury, sympathoadrenal activation, coagulation activation/inhibition and fibrinolysis were measured in uniplicate by commercially available immunoassays according to the manufactures recommendations. In each patient, a total of 15 biomarkers were measured corresponding to a total of 15*80 = 1,280 measurements, with only 3 missing measurements. The biomarkers were analyzed in EDTA or citrate plasma as follows: EDTA plasma: adrenaline and noradrenaline (2-CAT ELISA, Labor Diagnostica Nord GmbH & Co. KG, Nordhorn, Germany; lower limit of detection (LLD) 11 pg/ml (adrenaline, normal reference <100 pg/ml) and 44 pg/ml (noradrenaline, normal reference <600 pg/ml), respectively. Histone-complexed DNA fragments (hcDNA, Cell Death Detection ELISA PLUS , Roche, Hvidovre, Denmark; LLD not stated, relative quantification); D-dimer (ADI; LLD 2-4 ng/ml) and sCD40L (R&D Systems Europe; LLD 4.2 pg/ml). Citrate plasma: protein C (PC, Helena Laboratories, Beaumont, TX, US; LLD 5% of reference plasma); activated protein C (APC, USCNLIFE; LLD 4.2 pg/ml); tissue-type plasminogen activator (tPA, ADI, detects sc-tPA, tc- tPA and tPA/PAI-1 complexes; LLD 1 ng/ml); plasminogen activator inhibitor-1 (PAI-1, Assaypro; LLD 0.2 ng/ml); prothrombinfragment 1 and 2 (PF1.2, USCNLIFE; LLD 0.043 nmol/l); plasmin/α 2 -antiplasmin complex (PAP, ADI; LLD not stated); thrombin/antithrombin complex (TAT, USCNLIFE; LLD 0.215 ng/ml); tissue factor pathway inhibitor (TFPI, ADI, detects intact TFPI, truncated TFPI, TF/FVIIa/TFPI complexes; LLD 0.18 8 ng/ml); von Willebrand Factor antigen (vWF, Helena Laboratories, LLD 5% of reference plasma); factor XIII (FXIII, Assaypro; LLD 50 pg/ml). Thrombelastography (TEG) Whole blood clot formation was assessed in 3.2% citrated whole blood using a TEG® 5000 Hemostasis Analyzer System (Haemonetics Corp., MA, US), according to the manufacturers recommendations. All analyses were conducted within 2 hours from blood sampling at 37 °C. The variables recorded were [normal range reported by Haemonetics Corp.]: reaction time (R [3-8 min], rate of initial fibrin formation), angle (α [55-78 degrees], clot growth kinetics, reflecting the thrombin burst), maximum amplitude (MA, clot strength [51-69 mm], reflecting maximum clot strength) and lysis after 30 min (Ly30 [0-8 %], proportional reduction in the amplitude after MA, reflecting fibrinolysis) [5]. Patients were stratified according to TEG MA into the following groups: Normocoagulable (MA from 51-69 mm, n=69), hypercoagulable (MA >69 mm, n=9), hypocoagulable (MA <51, n=1) and hypocoagulable hyperfibrinolysis (MA <51 and Ly30 >8%, n=1). The day-to-day CV% of TEG MA is <7% in our laboratory [22]. Statistics Statistical analysis was performed using SAS 9.1 (SAS Institute Inc., Cary, NC, US). Data from patients stratified according to ISS group (ISS > 26, ISS 15-26, ISS <15) or maximum clot strength (TEG MA, normal vs. hypercoagulable) were compared by Kruskal-Wallis and Bonferroni adjusted Wilcoxon Rank Sum post hoc tests, Wilcoxon Rank Sum tests and Chi-square/Fischer exact tests, as appropriate. The contribution of platelets, fibrinogen, FXIII and biomarkers to the variation in maximum clot strength was investigated separately in each ISS group by univariate and multivariate linear regression analysis. Data are presented as medians with inter quartile ranges (IQR). P-values <0.05 were considered significant. 9 Results Study patients The 80 patients presented with ISS in the entire range (ISS >26 n=23, 15-26 n=26 and <15 n=30), with demography, injuries, transfusion requirements, mortality, biochemistry, thrombelastography and biomarkers as depicted in Table 1. Most patients (96%) were referred by mobile emergency care units (MECU) staffed with anesthetists (26% by helicopter) and blood samples were drawn a median of 68 min (IQR 48-88) after the injury. Increasing ISS was associated with higher mortality (18% overall mortality), lower Glascow Coma Score scale, increased volume of prehospital crystalloids and higher blood transfusion requirements, catecholamines, biomarkers of tissue injury and shock (pH, lactate, SBE) (Table 1). Mortality causes were in brief: Of the 11 patients whom expired in the group with normal TEG, eight died within 24h (50% from severe (s) TBI) and three died on days 7, 7 and 24 post-injury, two from sTBI sequels. The two patients who expired in the group with hypercoagulable TEG died days 7 and 8 post-injury, one from sTBI sequels. The patients with hyperfibrinolysis died within 24h from severe non-TBI injuries. Injury severity and coagulopathy ACT defined by APTT or INR above normal, were present in 15% of all patients (8% and 13% had increased APTT and INR, respectively) with increasing prevalence in the highest ISS group (Table 1). Furthermore, increasing ISS was associated with reduced fibrinogen and FXIII levels and also with reduced TEG R time and increased Ly30. With regards to biomarkers of coagulation activation, increasing ISS was associated with increased sCD40L, a biomarker of platelet activation, and with significantly increased PF1.2 in moderately injured patients (ISS 15- 26) as compared to both more severely and less injured patients. A similar tendency was observed for TAT (Table 1). Considering biomarkers of natural anticoagulation and fibrinolysis, AT and non-activated PC declined with increasing ISS whereas APC and D-dimer increased (Table 1). [...]... indicates that excessive sympathoadrenal activation and platelet activation in the most severely injured patients may contribute to the acute coagulopathy of trauma 15 Abbreviations α, TEG angle; ACIT, activation of coagulation and inflammation after trauma; ACT, acute coagulopathy of trauma; APC, activated protein C; APTT, activated partial thromboplastin time; ELISA, enzyme linked immunosorbent assay;... activated partial thromboplastin time; INR, international normalized ratio Biomarker abbreviations, see Materials and Methods section, ELISA 24 Table 2 Demography, injury severity, transfusion requirements, mortality, biochemistry and hemostasis, thrombelastography and biomarkers of coagulopathy in 80 trauma patients stratified according to TEG profile (normal, hypercoagulability, hypocoagulability, hyperfibrinolysis)... Johansson PI, Ostrowski SR: Acute coagulopathy of trauma: Balancing progressive catecholamine induced endothelial activation and damage by fluid phase anticoagulation Med Hypotheses 2010, 75:564-567 21 Davenport R, Manson J, De'ath H, Platton S, Coates A, Allard S, Hart D, Pearse R, Pasi KJ, Maccallum P, Stanworth S, Brohi K: Functional definition and characterization of acute traumatic coagulopathy. .. notable that the hypercoagulable and normal patients had comparable mortality despite a considerably higher age in the hypercoagulable group It is tempting to speculate that a hypercoagulable response to moderate (survivable) trauma may be optimal from an evolutionary perspective by promoting hemostasis Given this, the hypocoagulability and/ or hyperfibrinolysis that may accompany severe (unsurvivable)... interpretation of data, figure drafting and drafting/writing/revising of the manuscript AMS and CFL contributed to the design of the study and revised the manuscript critically PIJ contributed to the conception and design of the study, interpretation of data and drafting/writing/revising of the manuscript All authors read and approved the final manuscript Acknowledgements Karen Dyerermose and Marie Helena Andersson... Kluger Y, Mackway-Jones K, Parr MJ, Rizoli SB, Yukioka T, Hoyt DB, Bouillon B: The coagulopathy of trauma: a review of mechanisms J Trauma 2008, 65:748-754 4 Frith D, Brohi K: The acute coagulopathy of trauma shock: Clinical relevance Surgeon 2010, 8:159163 5 Johansson PI, Stissing T, Bochsen L, Ostrowski SR: Thrombelastography and tromboelastometry in assessing coagulopathy in trauma Scand J Trauma Resusc... respectively, and these are displayed separately in Table 2 for comparison, though no attempt was done to statistically compare these patients with the normal or hypercoagulable groups It is notable that fibrinogen, FXIII and thrombin generation (PF1.2) was profoundly reduced in these two patients and that adrenaline, hcDNA, sCD40L and tPA was markedly increased in the patient with primary hyperfibrinolysis... negatively independently associated with clot strength only in the highest ISS group indicating that excessive sympathoadrenal activation may negatively influence hemostasis We recently proposed [20] and demonstrated [18] that progressive increases in adrenaline levels in trauma patients promote a switch from hypercoagulability towards hypocoagulability and hyperfibrinolysis due to the influence of adrenaline... confirmed in a larger study powered to investigate biomarkers in patients with hypocoagulable or hyperfibrinolytic TEG profiles Increasing injury severity was associated with lower fibrinogen levels and importantly also with lower FXIII levels and higher prevalence of patients with ACT according to APTT or INR The association between injury severity and fibrinogen, APTT and INR is well established [3;4]... fibrinogen level and platelet count Furthermore, increasing injury severity was associated with increased shock, sympathoadrenal activation, tissue injury, platelet activation, protein C activation (higher activated PC, lower non-activated PC), hyperfibrinolysis and reduced fibrinogen and FXIII levels Finally, in the most severely injured patients (highest ISS group), adrenaline and sCD40L were independently . coagulopathy of trauma. 16 Abbreviations α, TEG angle; ACIT, activation of coagulation and inflammation after trauma; ACT, acute coagulopathy of trauma; APC, activated protein C; APTT, activated. TEG profiles. The primary aim of this study was to investigate biomarkers of sympathoadrenal activation, tissue injury, coagulation activation/inhibition and fibrinolysis in trauma patients. normal to hypercoagulability, hypocoagulability and hyperfibrinolysis) would be accompanied by evidence of increased sympathoadrenal activation, tissue injury, PC activation and hyperfibrinolysis